Traditional and Molecular Methods vs Biosensors for the Detection of Pathogens in Poultry Meat

The research of pathogenic microorganisms in foods has seen an intensification of the efforts to develop more specific and sensitive methods able to ascertain the presence of any virus or bacteria potentially harmful for health before marketing the products. This thesis was developed on the basis of these considerations. The purpose was to optimize molecular biology techniques that allow to reduce the time, often very long, required by traditional microbiology and to develop new based-biosensor protocols to get a fast and precise result. This is a very important aspect for food industries that need answers in a short time to avoid or limit economic losses. Campylobacter’s porA gene, 16S rRNA gene and 16S-ITS-23S operon (Chapter 2) were used for the direct detection of the pathogens found in poultry meat through the utilization of primers and probes annealing specific DNA sequences. The great genetic variability into the porA gene of different Campylobacter species was used for the design of four specie-specific sets of primers able to distinguish between the four most important pathogenic species in humans and animals. On the contrary, 16S rDNA gene (that codes for the rRNA component of the ribosomes) is a conserved section of prokaryotic DNA found in all bacteria and Archaea and it is a useful tool for studying bacterial communities. For this reason it has been choosen to design two probes for the quick identification at genus level with Dot Blot and OLED biochip. The sequence of the internal transcribed spacer (ITS) region between the 16S and 23S rRNA, variable in size depending from the species considered, was used to design a unique couple of primers that allows to differentiate, in a single PCR assay, between the three Campylobacter species mainly involved in foodborne disease. Diagnosis of Listeria monocytogenes (Chapter 3) is normally made through the use of internationally certified (ISO) methods which need the selective enrichment and the streak on selective and differential media. For this reason, as for Campylobacter, the aim of the work was the direct detection of the pathogen found in poultry meat through the utilization of primers and probes annealing specific DNA sequences. For the obtainement of the results, the probes and primers used were designed within a gene own of the genus Listeria, the iap gene, selecting portions of the sequence that ensure reliable identification for L. monocytogenes, as the only species pathogenic for humans. PCR and Dot Blot, used to confirm and optimize the identification obtained through the use of traditional ISO methods, and a preliminary study applying magnetic beads and LSPR biosensor protocols were made to verifiy the possibility of diagnosis of this pathogen through the use of these biosensors.